Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Jan 1;68(Pt 1):63-5. doi: 10.1107/S1744309111048111. Epub 2011 Dec 24.

Abstract

L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD(+). In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD(+) and the crystal diffracted to 2.38 Å resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 Å. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD(+), and data sets were collected to 2.20 and 2.49 Å resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 Å and a = b = 133.43, c = 99.09 Å, respectively. Tetramers were observed in the asymmetric units of all three crystals.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacillus subtilis / enzymology*
  • Crystallization
  • Crystallography, X-Ray
  • Gene Expression
  • L-Lactate Dehydrogenase / chemistry*
  • L-Lactate Dehydrogenase / genetics
  • L-Lactate Dehydrogenase / isolation & purification
  • Mutation*

Substances

  • L-Lactate Dehydrogenase